Host genetic risk factors to viral diseases -a double-edged sword

Linköping University Medical Dissertation No. 1183

Host genetic risk factors to viral diseases

-a double-edged sword

studies of norovirus and tick-borne encephalitis virus

Elin Kindberg

Department of Clinical and Experimental Medicine Linköping University, Sweden

© Elin Kindberg, 2010

Cover: illustration resembling virus particles as seen in an electron microscope. By Nina Frisk

Published articles have been reprinted with the permission of the copyright holder. Printed in Sweden by LiU-Tryck, Linköping, Sweden, 2010

ISBN 978-91-7393-393-3 ISSN 0345-0082

“Den vinner som är trägen, den förlorar som ger upp” Lars Winnerbäck, “Elden”

“A virus is a piece of bad news wrapped in protein” Sir Peter Medawar

Table of contents

ABSTRACT ... 1

PAPERS IN THE PRESENT THESIS ...3

ABBREVIATIONS ...5

INTRODUCTION ...7

Norovirus... 8

Tick-borne.encephalitis.virus...25

AIMS OF THE THESIS ...35

MATERIALS ...37 Paper.I...37 Paper.II...37 Paper.III...37 Paper.IV.and.V...37 METHODS ...39 Pyrosequencing.(Paper.I-V)... 39 ABO.blood.group.and.Lewis.a/b.antigen.ELISA.(Paper.II.and.III)...42 Statistical.calculations... 43

RESULTS AND DISCUSSION ... 45 Paper.I.–.Host.genetic.resistance.to.symptomatic.norovirus.(GGII.4).infections.in.Denmark... 45 Paper.II.–.The.G428A.nonsense.mutation.in. .provides.strong.but.not.absolute.protection.against. symptomatic.GII.4.norovirus.infection... 46 Paper.III.–.Norovirus.gastroenteritis.outbreak.with.a.secretor-independent.susceptibility.pattern,. Sweden... 48 Paper.IV.–.A.deletion.in.the.chemokine.receptor.5.( ).gene.is.associated.with.tick-borne. encephalitis... 50 Paper.V.–.A.missense.mutation.in.the.toll-like.receptor.3.gene.( ).is.associated.with.decreased. risk.of.tick-borne.encephalitis... 51 CONCLUSIONS ...55

FUTURE AND ONGOING STUDIES...57

POPULÄRVETENSKAPLIG SAMMANFATTNING ...59

ACKNOWLEDGEMENTS ... 61

Abstract

It.is.today.well.known.that.the.outcome.of.a.certain.infection.depends.on.factors.of.both. the.host.and.the.pathogen..Studies.of.host.genetic.susceptibility.to.infectious.diseases.aim. to.increase.the.understanding.of.why.some.individuals.are.more.susceptible.than.others,. to.a.certain.infection..Knowledge.of.genetic.susceptibility.to.a.viral.disease.may.be.used. in.development.of.new.therapeutic.means,.and.also.to.recognize.individuals.who.are.at. increased.risk.of.severe.symptoms.if.infected.with.a.pathogen..It.seems.however.that.a.risk. factor.for.one.disease.may.play.a.protective.role.in.another.situation;.like.a.double-edged. sword... In.this.thesis.I.have.studied.genetic.factors.affecting.susceptibility.to.norovirus.(NoV).and. factors.affecting.the.risk.of.developing.tick-borne.encephalitis.(TBE).after.infection.with.TBE. virus.(TBEV)..NoV.is.the.cause.of.the.“winter.vomiting.disease”,.affecting.millions.of.people. every.year,.and.causing.up.to.200,000.fatalities.among.children.in.developing.countries,. each.year..It.is.today.recognized.that.the.secretor.status.of.an.individual,.i.e..the.ability. to.express.ABO.blood.groups.and.related.antigens,.in.secretions.and.on.mucosa,.affect. the.risk.of.being.infected.by.NoV..By.studying.authentic.NoV.outbreaks.in.Denmark,.Spain. and.Sweden.and.by.comparing.the.secretor.status.of.affected.and.unaffected.individuals. we.were.able.to.confirm.that.secretor.status.have.indeed.great.impact.on.susceptibility. to.some. NoV.strains,.but.also.that.there. are. strains.circulating,.which.infect.individuals. regardless.of.secretor.status. TBEV.is.endemic.in.many.parts.of.Europe.and.Asia.but.studies.have.shown.that.70-95%.of. all.infections.are.asymptomatic.or.sub-clinical..Some.individuals.do.however.develop.TBE,. a.severe.disease.including.meningitis.or.encephalitis.with.or.without.myelitis..Also,.many. patients.suffer.from.long-time.sequelae.and.TBEV.infections.may.in.worst.case.be.fatal.. The.reason.for.difference.in.disease.outcome.is.not.known.and.we.have.chosen.to.study.if. genetic.factors.affecting.the.immune.response.may.play.a.role.in.disease.outcome..To.do. this.we.used.a.prospectively.collected.Lithuanian.material.with.samples.from.patients.with. TBE,. AME. (aseptic. meningoencephalitis).and. matched. healthy. controls.. So. far. we. have. found.that.a.deletion.in. .( ),.a.gene.encoding.a.receptor.involved. in.cell.migration,.is.a.risk.factor.for.developing.disease..We.have.also.data.showing.that. toll-like.receptor.3.(TLR3),.a.receptor.recognizing.double.stranded.RNA.(dsRNA),.which.is.a. product.of.TBEV.replication,.may.instead.of.being.protective.increase.the.risk.of.TBE..

Papers in the present thesis

The.papers.will.be.referred.to.by.their.roman.numerals

I.. Host genetic resistance to symptomatic norovirus (GGII.4) infections in Denmark.

Kindberg.E,.Åkerlind.B,.Johnsen.C,.Knudsen.JD,.Heltberg.O,.Larson.G,.. . . Böttiger.B,.Svensson.L..J.Clin.Microbiol..2007;45(8):2720-2

II.. The G428A nonsense mutation in FUT2 provides strong but not absolute protection against symptomatic GII.4 norovirus infection.

. Carlsson.B*,.Kindberg.E*,.Buesa.J,.Rydell.GE,.Lidón.MF,.Montava... . . R,.Abu.Mallouh.R,.Grahn.A,.Rodríguez-Díaz.J,.Bellido.J,.Arnedo.A,.Larson.G,.. . Svensson.L..PLoS.One..2009;4(5):e5593.

. *Contributed.equally.to.this.work.

III. Norovirus gastroenteritis outbreak with a secretor-independent susceptibility pattern, Sweden.

. Nordgren.J,.Kindberg.E,.Lindgren.PE,.Matussek.A,.Svensson.L..Emerg.Infect.. . Dis..2010;16(1):81-7.

IV.. A deletion in the chemokine receptor 5 (CCR5) gene is associated with tick-borne encephalitis.

Kindberg.E,.Mickiene.A,.Ax.C,.Åkerlind.B,.Vene.S,.Lindquist.L,.Lundkvist.Å,.. . Svensson.L..J.Infect.Dis..2008;197(2):266-9.

V.. A missense mutation in the toll-like receptor 3 gene (TLR3) is associated with decreased risk of tick-borne encephalitis.

Kindberg.E,.Vene.S,.Mickiene.A,.Lundkvist.Å,.Lindquist.L,.Svensson.L,.. . . Manuscript

Abbreviations

3CLPro. 3C-like.protease aa.. . amino.acid(s) ab/abs. antibody/antibodies AGE. . acute.gastroenteritis ATP. . adenosine.triphosphate bbb. . blood-brain.barrier CCR5.. chemokine.receptor.5.gene/protein cDNA. . complimentary.DNA CSF. . cerebrospinal.fluid DNA. . deoxyribonucleic.acid dsRNA. double.stranded.RNA ELISA. . enzyme-linked.immunosorbent.assay EM. . electron.microscopy Fuc. . fucose FucT-II. α1,2-fucosyltransferase.2 FucT-III. α1,3/4-fucosyltransferase.3 . . fucosyltransferase.2,.the.gene.encoding.FucT-II fucosyltransferase.3.(Lewis.gene).encoding.FucT-III GI,.GII.. genogroup.I,.genogroup.II Gal. . galactose GalNAc. -acetylgalactosamine Glc. . glucose GlcNAc. -acetylglucosamine HBGA... histo-blood.group.antigen kb. . kilobases KO. . knock.out Lea_,.Leb_{.. Lewis.a.antigen,.Lewis.b.antigen} mut. . mutant NoV. . norovirus nt.. . nucleotide OAS. . 2’-5’-oligoadenylate.synthetase. OR. . odds.ratio ORF. . open.reading.frame PCR. . polymerase.chain.reaction PPi. . pyrophosphate.. RdRp. . RNA-dependent.RNA-polymerase RNA. . ribonucleic.acid RT. . reverse.transcriptase

SMV. . Snow.Mountain.virus SNP.. . single.nucleotide.polymorphism ssDNA. single.stranded.DNA ssRNA.. single.stranded.RNA TBE. . tick-borne.encephalitis TBEV. . tick-borne.encephalitis.virus TLR.. . toll-like.receptor VLP. . virus-like.particle VPg. . viral.protein.genome-linked WNV. . West.Nile.virus wt. . wild-type

Introduction

It.was.early.noted.that.the.outcome.of.a.certain.infection.differed.between.individuals. (Allison.1954;.Miller.et.al..1976;.Frodsham.and.Hill.2004;.Casanova.and.Abel.2007)..It. was.however.long.thought.that.these.variations.were.due.to.differences.associated. with.the.pathogen.rather.than.the.patient..Today.it.is.well.known.that.host.factors.can. have.large.impact.on.susceptibility.to.disease.(Alcais.et.al..2009).and.these.factors.can. be.of.genetic.or.non-genetic.kind..Age,.nutritional.status,.other.underlying.diseases. and.medication.are.non-genetic.factors.that.may.have.an.impact.on.the.risk.of.being. infected. with. a. microorganism..Genetic. factors. can. be. up. or. down. regulation.of. cytokines,.antibody.deficiencies,.complement.deficiencies.and.many.more.factors.of. immunologic.nature..It.can.also.be.low.levels.or.complete.absence.of.a.viral.receptor. The.human.immune.system.is.strong.at.population.level.but.weak.at.the.individual. level. (Casanova. and. Abel. 2005).. Together. we. can. protect. our. species. against. most.–.if.not.all.–.pathogens.but.each.of.us.is.only.protected.against.a.subset.of. the. microorganisms. circulating. in. the. environment.. While. some. individuals. will. be. asymptomatically. infected,. if. infected. at. all. by. a. bacteria. or. virus,. others. will. develop. severe. disease. or. even. die.. The. studies. of. host. genetic. susceptibility. to. infectious.disease.aim.to.increase.the.understanding.of.why.some.individuals.are. more.susceptible.than.others,.to.a.certain.infection.. On.several.occasions,.large.groups.of.individuals.have.by.mistake.been.exposed.to. infectious.agents,.for.example.via.contamined.blood.products.or.vaccines..In.1927,. 251.newborns.were.by.mistake.vaccinated.with.virulent. . . and.77.of.these.died.from.the.infection.(Murray.2004)..In.the.1970’s.hepatitis.C.virus. (HCV).contaminated.anti-rhesus.D.immunoglobulin.was.given.to.over.60,000.rhesus-negative.women.in.Ireland,.of.which.704.were.later.showing.signs.of.past.or.current. HCV.infection.(Kenny-Walsh.1999)..Furthermore,.in.the.1980’s.hemophiliacs.were. given.blood.products.contaminated.with.human.immunodeficiency.virus.(HIV).and. it.was.early.noted.that.the.disease.progression.rate.differed.between.the.infected. recipients.(O’Brien.and.Nelson.2004)..These.are.all.examples.of.where.a.large.group. of.people.have.been.infected.by.a.pathogen.but.with.very.different.outcome.. In. this. thesis. I. discuss. how. genetic. differences. between. individuals. affect. our. susceptibility.to.viral.disease,.more.precisely.to.infection.with.norovirus.(NoV).and. to. development. of. tick-borne. encephalitis. (TBE). after. infection. with. tick-borne. encephalitis.virus.(TBEV).

Norovirus

NoV.is.the.most.common.cause.of.viral.gastroenteritis.worldwide.(Atmar.and.Estes. 2006)..According.to.the.world.health.organization.(WHO),.18-19%.of.deaths.among. children.less.then.5.years.of.age.are.due.to.diarrhea,.meaning.that.almost.2.million. children.die.every.year.due.to.diarrheal.disease.(Bryce.et.al..2005;.Boschi-Pinto.et.al.. 2008)..NoV.has.been.associated.with.12%.of.all.severe.gastroenteritis.cases.among. children.<5.years.of.age.and.with.12%.of.mild.and.moderate.diarrhea.cases.of.all. ages.(Patel.et.al..2008)..It.has.been.estimated.to.cause.up.to.23.million.episodes.of. acute.gastroenteritis.(AGE).annually.in.the.US.(Mead.et.al..1999).and.to.cause.up.to. 200,000.fatalities.each.year,.among.children.<5.years.of.age.in.developing.countries. (Patel.et.al..2008)..

Already. in. 1929,. Zahorsky. described. the. phenomenon. of. increased. incidence. of. AGE.during.wintertime.and.hence.suggested.the.name.“winter.vomiting.disease”. (Zahorsky. 1929).. It. was. however. not. until. 1972. that. Albert. Kapikian,. by. electron. microscopy.(EM).(Figure.1),.discovered.NoV.as.the.cause.of.the.disease.(Kapikian.et. al..1972),.still.making.it.the.first.virus.to.be.associated.to.AGE..The.prototype.NoV.is. called.Norwalk.virus,.after.the.place.where.it.was.first.found,.in.a.school.outbreak.in. Norwalk,.Ohio.in.the.US.(Adler.and.Zickl.1969).and.the.genus.NoV.was.earlier.called. Norwalk-like.virus.after.the.prototype..NoV.are.small.(approximately.30.nm),.non-enveloped,.icosahedral.viruses.with.a.T=3.structure.and.single.stranded.ribonucleic. acid.(ssRNA).genome.of.approximately.7.7.kilobases.(kb).(Figure.2.and.3).(Green.et. al..2001)..The.virus.was.first.classified.by.its.appearance,.as.“small.round.structured. viruses”.(SRSVs).(Green.et.al..2001),.but.cloning.and.sequencing.later.classified.the. genus.as.a.calicivirus.(Xi.et.al..1990).. Figure.1..Clinical.sample.of.NoV.as.seen.in.transmission.electron.microscopy..The.virus.is.visualized. by.negative.staining.using.phosphotungstic.acid.

Genome structure and norovirus classification

NoV’s.positive-stranded.RNA.has.a.5´-VPg.(a.genome-linked.viral.protein).functioning. as.a.cap-substitute.(Daughenbaugh.et.al..2003;.Goodfellow.et.al..2005).and.a.3´-poly. A.tail.(Green.et.al..2001)..The.RNA.has.three.open.reading.frames.(ORF).(Green.et. al..2001)..ORF1.encodes.non-structural.proteins.such.as.the.RNA-dependent.RNA-polymerase. (RdRp). and. the. 3CLpro. protease. (Figure. 3). (Hardy. 2005). and. ORF2. encodes.the.large.capsid.protein.(Jiang.et.al..1992),.and.consist.of.an.S-region.and. the.P1.and.P2.regions..The.S.region.encodes.the.inner.“shell”.part.of.the.capsid. while.P1.and.P2.encodes.the.more.protruding.parts..P2.is.believed.to.take.part.in. the.interaction.with.receptor.structures.of.the.target.cells,.although.much.is.still. unknown.regarding.binding.sites.and.the.true.viral.receptor/s.for.NoV. Figure.2..Schematic.overview.of.the.NoV.particle..NoV.are.non-enveloped.and.contain.two.structural. proteins,. VP1. (the. capsid). and. the. minor. VP2.. (Viralzone,. http://www.expasy.ch/viralzone. Swiss. Institute.of.Bioinformatics,.with.permission).

Figure. 3.. Organization. of. the. NoVs. genome.. The. genome. is. a. ssRNA. (approximately. 7.7. kb). with. positive. polarity,. and. three. open. reading. frames. (ORF1-3).. The. RNA. genome. carries. a. 3´-poly.A.tail.and.a.5´-VPg,.the.latter.which.may.play.a.role.both.during.replication.and.translation. (Daughenbaugh.et.al..2003;.Mitra.et.al..2004;.Goodfellow.et.al..2005;.Rohayem.et.al..2006)..ORF1. encodes.a.polyprotein.which.is.cleaved.by.the.viral.protease.3CLpro.into.at.least.six.non-structural. proteins,.such.as.the.polymerase.(RdRp).and.the.NTPase,.ORF2.encodes.the.capsid.gene.with.an. N-terminal.domain.(N).the.inner.shell.(S).and.outer.protruding.(P).parts.and.ORF3.encodes.a.minor. structural.protein,.VP2.which.may.have.a.stabilizing.affect.on.the.viral.capsid.(Bertolotti-Ciarlet.et. al..2003;.Patel.et.al..2008).

NoV.is.a.genus.within.the.family. (Figure.4)..Other.genera.of.this.family. are.sapovirus,.vesivirus,.lagovirus.and.nebovirus.(Thiel.and.Konig.1999;.Oliver.et.al.. 2006)..The.genus.lagovirus.includes.the.rabbit.hemorrhagic.disease.virus.(RHDV),.a. virus.causing.severe.infections.in.rabbits,.including.enlargement.of.the.liver.and.a. necrotizing.hepatitis.(Thiel.and.Konig.1999).often.with.a.fatal.outcome.(Ohlinger.et. al..1990)..Vesicular.exanthema.of.swine.virus.(VESV).and.feline.calicivirus.(FCV).are. virus.in.the.vesivirus.genus,.infecting.pigs.and.cats,.respectively.(Thiel.and.Konig. 1999)..Nebovirus.(Oliver.et.al..2006).cause.gastroenteric.symptoms.in.cattle.(Woode. and.Bridger.1978;.Bridger.et.al..1984)..Sapovirus,.as.NoV,.cause.AGE.in.humans.but.is. a.far.less.common.cause.than.NoV.of.the.disease.(Hedlund.et.al..2000;.Green.et.al.. 2001;.Moreno-Espinosa.et.al..2004).. Figure.4..Schematic.drawing.of.the. .family..Bold.lining.indicates.pathogens.discussed.in. this.thesis.. .consists.of.five.genera.of.which.NoV.and.sapovirus.infect.humans..NoV.GI. and.GII.are.the.most.common.genotypes.infecting.humans.although.NoV.GIV.may.also.be.found.in. human.AGE.cases..NoV.GII.can.also.infect.pigs.(Sugieda.and.Nakajima.2002).and.GIV.has.been.found. in.dogs.and.lions.(Martella.et.al..2007;.Martella.et.al..2008).

The. genus. NoV. is. divided. into. five. genogroups. (GI-V). (Figure. 4). and. further. into. genotypes. depending. on. similarity. of. the. nucleotide. (nt). sequence. of. the. polymerase.or.capsid.genes.(Figure.3)..The.nt.similarity.between.strains.within.a. genotype.is.69–97%,.while.the.similarity.between.strains.of.different.genogroups.is. 51–56%.nt.(Katayama.et.al..2002)..Comparing.the.amino.acid.(aa).sequence.of.the. major.structural.NoV.protein,.the.difference.within.a.genotype.can.be.up.to.40%. and.between.genogroups.the.difference.is.generally.more.than.50%.(Pletneva.et.al.. 2001).. GI,.GII.and.GIV.NoV.infect.humans,.GIII.are.bovine.NoV.and.GV.infects.mice..GII.NoV. have.also.been.detected.in.fecal.samples.from.healthy.pigs.(Sugieda.and.Nakajima.

2002). and. GIV. NoV. has. been. detected. in. lions. (Martella. et. al.. 2007). and. dogs. (Martella.et.al..2008)..To.date,.at.least.8.GI.and.17.GII.clusters.have.been.detected. (Zheng.et.al..2006)..Although.NoV.infects.both.humans.and.animals,.no.cross-species. transmission.of.strains.have.been.detected.so.far.(Atmar.and.Estes.2006)..However,. few.studies.have.shown.that.humans.can.have.antibodies.(abs).to.bovine.GIII.NoV. (Widdowson.et.al..2005b;.Vildevall.et.al..2010).and.also.that.gnotobiotic.calves.and. pigs.can.develop.symptoms.after.inoculation.with.human.GII.4.NoV.(Cheetham.et. al..2006;.Souza.et.al..2008)..Together,.these.data.suggest.that.zoonotic.spread.may. occur,.but.the.importance.of.this.remains.to.be.elucidated.. While.GI,.GII.and.GIV.NoV.can.all.cause.AGE.in.humans,.GI.and.particularly.GII.are. the.genotypes.most.commonly.involved.in.human.NoV.outbreaks..GII.4.has.been. dominating. globally. since. the. mid. 1990’s. (Noel. et. al.. 1999;. Lopman. et. al.. 2004a;. Widdowson. et. al.. 2004). and. has. been. estimated. to. cause. up. to. 80%. of. all. NoV. outbreaks.(Ramirez.et.al..2009).. New.GII.4. variants.have.been.detected. in.1996,. 2002,.2004.and.2006.(Bull.et.al..2006;.Siebenga.et.al..2007a)..GII.4.seems.to.dominate. especially.in.outbreaks.occurring.in.closed.settings.(Noel.et.al..1999;.Lindesmith.et. al..2008;.Fukuda.et.al..2009;.Siebenga.et.al..2009)..A.study.of.Nicaraguan.children. have.shown.that.GII.4.strains.are.shed.in.higher.titers.than.other.strains.(Bucardo. et.al..2008),.which.may.increase.the.ability.of.these.strains.to.spread.to.new.host.. Furthermore,.as.described.later.(see.“Immunity.and.viral.evolution”),.GII.4.strains. seems.to.be.able.to.escape.herd.immunity.by.epochal.evolution.(Siebenga.et.al.. 2007b;.Lindesmith.et.al..2008),.which.may.increase.its.ability.to.stay.in.the.population..

Symptoms and diagnosis

The.general.symptoms.of.NoV.disease.are.nausea,.vomiting.and.diarrhea,.sometimes. accompanied.by.low-grade.fever,.headache.and.joint.and.muscle.pain.(Green.et.al.. 2001;.Donaldson.et.al..2008;.Halperin.et.al..2008;.Nordgren.et.al..2010)..The.incubation. time.for.a.NoV.infection.is.generally.1-2.days.(Estes.et.al..2006).and.the.disease.is. generally.self-limiting..NoV.infections.have.on.rare.occasions.been.associated.with. adverse.effects.such.as.acute.renal.failure.(Kanai.et.al..2010),.encephalopathy.(Ito. et.al..2006).and.disseminated.intravascular.coagulation,.obtundation,.headache.and. photophobia.(CDC..2002).and.few.cases.of.viremia.have.been.reported.(Takanashi. et.al..2009;.Medici.et.al..2010)..A.recent.study.also.described.an.association.between. NoV.infection.and.development.of.two.cases.of.urticaria.(Leiste.et.al..2008).. NoV.infections.can.be.diagnosed.by.various.methods..EM.or.immuno.EM.may.be. used.but.demands.high.concentrations.of.virus.(106_{.viral.particles/ml).and.expensive.} equipment,.as.well.as.highly.skilled.microscopists.(Atmar.and.Estes.2001),.making.

it.an.inconvenient.method.for.screening.of.large.materials..ELISA.(Enzyme-linked. immunosorbent.assay).is.a.quick.assay.that.can.differ.between.GI.and.GII.NoV.but.the. drawback.is.the.sensitivity.as.shown.in.comparisons.between.ELISA.and.polymerase. chain.reaction.(PCR).based.methods.(Atmar.and.Estes.2001;.Nordgren.et.al..2008).. PCR.based.methods.are.commonly.used,.as.they.are.sensitive.and.broadly.reactive,. when.using.primer.pairs.specific.for.GI.or.GII.NoV..RT-PCR.(reverse.transcriptase-PCR).is.used.worldwide.and.is.a.good.alternative.in.the.absence.of.broadly.reactive. enzyme. immunoassays. (Atmar. and. Estes. 2001).. By. using. quantitative. PCR,. the. amount.of.virus.can.be.determined,.giving.an.extra.dimension.to.the.test..By.using. quantitative.PCR.with.melting.curves,.the.genotype.(GI.or.GII).and.amount.of.NoV. RNA.can.be.determined.in.one.single.assay.(Nordgren.et.al..2008)..However,.while. PCR.is.a.sensitive.method.it.does.not,.in.contrast.to.EM,.confirm.the.presence.of. intact.viral.particles,.nor.can.it.detect.new.or.unknown.virus.. In.the.absence.of.a.good.detection.method.for.viral.pathogens,.the.so-called.Kaplan. critera.may.be.used.to.suggest.NoV.as.the.cause.of.non-bacterial.AGE..The.criteria. are.summarized.in.Table.1.and.were.proposed.by.Kaplan.and.coworkers.(1982).in.a. time.when.specific.NoV.detection.methods.were.absent..Recent.evaluation.of.the. criteria.have.suggested.them.useful.also.today,.when.used.as.a.whole.set.and.not.as. individual.components.(Turcios.et.al..2006).. Table.1.Kaplan.criteria.for.identification.of.NoV.as.the.cause.of.non-bacterial. gastroenteritis.outbreaks Stool.culture.negative.for.bacterial.pathogens Vomiting.in.at.least.50%.of.cases Mean/Median.incubation.time.24-48h Mean/Median.duration.of.symptoms.12-60h

Transmission and physical properties

NoV.is.spread.by.the.fecal-oral.route.and.transmission.generally.occurs.from.person-to-person. and. via. contaminated. food. or. water. (Green. et. al.. 2001),. even. though. airborne.transmission.may.also.occur.(Sawyer.et.al..1988;.Caul.1994;.Marks.et.al.. 2000;.Evans.et.al..2002;.Marks.et.al..2003;.Widdowson.et.al..2005a;.Atmar.and.Estes. 2006;. Kuo. et. al.. 2009).. There. is. a. high. rate. of. secondary. attacks. by. contacts. of. the.infected.people.(often.>30%),.which.often.lead.to.high.attack.rates.in.closed. settings.such.as.elderly.nursing.homes,.day.care.settings,.cruise.ships.and.hospital. wards.and.hotels.(Cheesbrough.et.al..2000;.Fankhauser.et.al..2002;.Isakbaeva.et.al.. 2005;.Kindberg.et.al..2007;.Michel.et.al..2007;.Carlsson.et.al..2009)..While.an.early. volunteer.study.suggested.that.NoV.is.shed.only.the.first.few.days.after.the.onset. of.the.symptoms.(Gary.et.al..1987),.more.recent.studies.have.shown.that.prolonged.

shedding.may.occur.(Nilsson.et.al..2003;.Kirkwood.and.Streitberg.2008;.Ludwig.et. al..2008;.Siebenga.et.al..2008)..Furthermore,.chronic.infections.have.been.seen.in. immuno-compromised.individuals.(Nilsson.et.al..2003;.Gallimore.et.al..2004b;.Ludwig. et.al..2008).and.it.has.been.shown.that.chronic.NoV.carriers.may.spread.infectious. virus.over.a.long.period.of.time.(Nilsson.et.al..2003)..Furthermore,.asymptomatic. NoV.infection.occurs.(Keswick.et.al..1985;.Johnson.et.al..1990;.Gallimore.et.al..2004a;. Ozawa.et.al..2007;.Atmar.et.al..2008;.Bucardo.et.al..2010).and.may.be.a.source.of. spread.. NoV.are.very.stable.and.studies.with.the.surrogate.murine.NoV.and.feline.calicivirus. have.shown.persistence.of.infectivity.after.treatment.with.a.range.of.pH.of.at.least. 3-9.and.a.temperature.up.to.56°.(Cannon.et.al..2006)..Moreover,.studies.using.feline. calicivirus.have.shown.that.70%.ethanol.is.not.sufficient.to.inactivate.the.virus.(Duizer. et.al..2004a)..A.study.using.human.NoV.have.also.shown.that.virus.is.continuously. spread. via. contaminated. hands,. contact. surfaces. and. cloths.. Furthermore,. hypochlorite.bleach.is.not.always.enough.to.eliminate.NoV..(Barker.et.al..2004)

Replication and pathogenesis

Despite.many.attempts,.no.convenient.cell-culture.or.animal.model.is.yet.present. for. human. NoV. (Duizer. et. al.. 2004b).. Asanaka. and. coworkers. (2005). and. Guix. and.coworkers.(2007).have.described.models.to.produce.viral.particles.from.NoV. complimentary. deoxyribonucleic. acid. (cDNA). and. RNA. respectively,. but. a. full. replication. cycle. could. not. be. presented.. Straub. and. coworkers. (2007). used. a. human.3D.tissue.culture.model.to.cultivate.NoV.and.was.able.to.cultivate.the.virus. over.several.passages,.accompanied.by.cytopathic.effects.such.as.shortening.and. vacuolization.of.the.villi..The.method.is.however.technically.challenging,.and.it.is. still.unclear.why.this.method.could.support.NoV.cultivation.while.other.methods. have.failed.(Vashist.et.al..2009)..The.lack.of.a.good.model.system.has.hampered.the. studies.of.NoV.pathogenesis.and.pathophysiology..Murine.NoV.may.be.cultivated. using.murine.macrophages.and.this.model.may.be.used.to.understand.the.replication. and.pathogenesis.of.NoV.(Wobus.et.al..2006).as.human.and.murine.NoV.are.closely. related.. Murine. NoV. does. however. not. cause. gastroenteric. symptoms. in. mice. but.rather.a.systemic.disease.in.STAT.deficient.mice.(Karst.et.al..2003)..All.these. observations.support.that.a.good.model.for.studying.the.pathophysiology.behind. NoV.diarrhea.and.vomiting.is.still.needed.. Diarrhea.is.the.result.of.rapid.movement.of.stool.through.the.large.intestine.(Guyton. and.Hall.2000)..There.are.different.causes.of.diarrhea;.an.influx.of.anions.into.the. lumen.causes.secretory.diarrhea,.an.impaired.digestion.of.osmotically.active.agents.

such.as.disaccharides.causes.osmotic.diarrhea.(Baldi.et.al..2009).and.a.disturbed. motility.of.the.intestine.causes.functional.bowel.disorders.such.as.irritable.bowel. syndrome.(Grundmann.and.Yoon.2010)..Diarrhea.may.also.be.caused.by.intestinal. inflammation.as.in.Chron’s.disease.and.in.ulcerative.colitis.(Binder.2009).or.by.nervous. tension. (Guyton. and. Hall. 2000).. The. exact. mechanisms. behind. NoV. diarrhea. are. still.not.known.but.information.about.NoV.pathogenesis.and.pathophysiology.from. volunteer.studies.(Dolin.et.al..1971;.Agus.et.al..1973;.Schreiber.et.al..1973;.Schreiber. et.al..1974;.Wyatt.et.al..1974;.Widerlite.et.al..1975;.Levy.et.al..1976;.Parrino.et.al.. 1977;.Meeroff.et.al..1980).and.authentic.cases.(Troeger.et.al..2008).have.given.some. possible.answers.to.the.question..The.target.cell.for.NoV.is.not.clearly.identified.but. viral.replication.is.thought.to.occur.in.the.upper.part.the.small.intestine,.in.duodenum. and.jejunum.(Agus.et.al..1973;.Schreiber.et.al..1973;.Schreiber.et.al..1974;.Troeger. et.al..2008)..Marionneau.and.coworkers.(2002).used.intestinal.tissue.sections.and. NoV. virus-like. particles. (VLPs),. to. study. viral. binding. and. showed. that. NoV. bind. mainly.to.the.intestinal.villi.and.less.to.cells.in.the.crypts..Volunteer.and.authentic. studies.on.human.NoV.pathogenesis.suggest.that.acute.NoV.infection.cause.only. limited.histo-pathological.lesions.in.duodenum.and.jejunum.with.broadening.and. blunting.of.the.villi,.in.parallel.with.the.appearance.of.immune.cell.infiltration.and. cytoplasmic.vacuolization.(Schreiber.et.al..1973;.Schreiber.et.al..1974;.Dolin.et.al.. 1975)..Adenylate.cyclase.seems.not.to.be.increased.during.NoV.infection.(Levy.et. al..1976)..Studies.of.duodenal.biopsies.from.patients.in.authentic.infections.have. shown.that.the.mononuclear.cells.infiltrating.the.villi.are.CD8+.T.cells.(Troeger.et.al.. 2008)..The.mechanism.of.rapid.atrophy.of.the.villi.may.hence.depend.on.activation. of. intraepithelial. lymphocytes. with. cytokine. mediated. or. cytotoxic. effect. on. enterocytes..Troeger.and.co-workers.(2008).also.showed.that.NoV.infection.causes. severe. epithelial. barrier. dysfunction. with. leakage,. and. an. active. anion. secretion. followed.by.passive.water.secretion..Blacklow.and.co-workers.(1972).reported.that. six.out.of.seven.volunteers.studied.showed.an.abnormal.absorption.of.D-xylose.and. decreased.ability.to.break.down.lactase.as.well.as.an.increased.excretion.of.fat.. Apart.from.diarrhea,.vomiting.is.a.common.symptom.of.NoV.infection..Vomiting.is. a.biologic.defense.mechanism.and.is.caused.by.stimuli.of.the.vomiting.center.in.the. medulla.(Guyton.and.Hall.2000)..Stimuli.can.be.transmitted.for.example.via.cranial. nerve.X,.the.vagus.nerve.(Blackshaw.et.al..2007)..The.stimulus.can.be.for.example. toxins,. gastritis,. chemotherapy,. motion. sickness. or. hormones. (Guyton. and. Hall. 2000)..Early.volunteer.studies.have.shown.that.no.histological.lesions.are.observed. in.the.gastric.fundus.and.antrum.of.volunteers.with.Norwalk.virus-induced.illness. (Widerlite.et.al..1975)..Gastric.secretion.of.hydrochloric.acid,.pepsin,.and.intrinsic. factors.appear.unaltered.during.infection.(Meeroff.et.al..1980)..While.the.gastric.

mucosa.remains.unaffected,.a.delay.in.gastric.emptying.was.observed.in.infected. volunteers.who.became.ill.and.developed.the.typical.jejunal.mucosal.lesions.(Meeroff. et.al..1980)..It.has.been.proposed.that.normal.gastric.motor.function.is.responsible. for.nausea.and.vomiting,.suggesting.a.neurogenic.mechanism.for.delayed.gastric. emptying..Thus.it.is.reasonable.to.believe.that.NoV,.similar.to.rotavirus,.activates. the.enteric.nervous.system.(Lundgren.et.al..2000;.Kordasti.et.al..2004).including. serotonergic.afferents.and.the.vagus.nerve,.to.trigger.the.vomiting.centre.in.the. central.nervous.system.(CNS).. Epidemiology

NoV. infections. occur. all. year. around,. but. as. the. name. winter. vomiting. disease. implies,.with.a.peak.incidence.during.the.wintertime.(Figure.5).(Hedlund.et.al..2000;. Mounts.et.al..2000)..An.analysis.of.twelve.studies.of.NoV.seasonality.showed.that. in.all.cases,.the.incidence.of.NoV.was.lower.during.warmer.than.colder.months,. with. no. difference. between. outbreaks. and. sporadic. cases. (Mounts. et. al.. 2000).. By.investigating.factors.of.the.host,.environment.and.the.virus,.Lopman.and.co-workers.(2009).conclude.that.several.factors.are.involved.in.the.development.of.a. new.NoV.peak..A.new.peak.in.incidence.occurs.during.cold,.dry.weather,.when.the. immunity.in.population.is.low.and.when.a.new.GII.4.strain.has.emerged..In.another. study,.Lopman.and.co-workers.(2003a).show.that.winter-seasonality.is.mainly.seen. for.outbreaks.in.health-care.institutions,.while.outbreaks.in.other.settings,.such.as. schools.and.hotels,.occur.at.an.even.rate.all.year.around..On.rare.occasion,.summer-time.peaks.of.NoV.infections.do.occur.(Lopman.et.al..2003b).

Figure. 5.. Weekly. record. of. NoV. cases. in. Sweden,. reported. to. the. Swedish. center. of. infectious. disease.control.(SMI)..(Data.from.K-O.Hedlund,.March.26,.2010).

Volunteer studies of norovirus susceptibility It.was.early.noted.that.susceptibility.to.symptomatic.NoV.infection.differed.between. individuals.(Parrino.et.al..1977;.Gary.et.al..1987;.Lindesmith.et.al..2003)..Parrino.and. coworkers.(1977).challenged.12.volunteers.with.the.GI.1.Norwalk.isolate.8FIIa.and. reported.that.only.6/12.individuals.developed.disease..After.a.second.challenge.27-42.months.later,.with.the.same.isolate,.the.same.six.individuals.showed.symptoms. of.NoV.disease..At.a.third.challenge.of.four.of.the.six.symptomatic.individuals,.4-8. weeks. after. the. second. challenge,. one. out. of. four. individuals. was. symptomatic,. showing. that. even. though. long-term. protection. seems. absent,. patients. may. be. protected. from. re-infection. shortly. after. the. previous. inoculation.. Furthermore,. investigation. of. antibody. (ab). titers. of. eight. participants. in. the. study. revealed. that.five.of.the.symptomatic.volunteers.had.high.ab.titers.to.NoV.while.none.of. three. asymptomatic. volunteers. had. high. anti-NoV. ab. titers,. neither. before. nor. after.challenge..This.suggests.that.lack.of.symptoms.was.not.correlated.to.a.strong. ab. response.. Interestingly,. already. at. this. time-point,. the. authors. discussed. the. possibility.of.a.genetic.factor.protecting.a.part.of.the.population.from.NoV.disease,. with.a.reference.to.the.recently.found.host.genetic.resistance.to.malaria.

In. 1990,. Johnson. and. coworkers. (1990). performed. a. study. using. 42. volunteers. and. found. an. association. between. high. pre-existing. ab. titers. and. increased. risk. of. symptomatic. GI.1. NoV. infection.. They. also. noted. that. a. short-term. immunity. developed. after. challenge.. Graham. and. coworkers. (1994). studied. symptomatic. and.asymptomatic.NoV.infection.after.infection.with.GI.1.and.saw.no.difference.in. susceptibility.between.individuals.with.or.without.pre-existing.abs,.and.also.that. NoV.was.still.present.in.stool.samples.collected.seven.days.after.inoculation.. Using.sera.from.volunteer.studies.used.in.an.earlier.study.(Graham.et.al..1994).Hutson. and. coworkers. (2002). studied. susceptibility. to. symptomatic. and. asymptomatic. GI.1.NoV.infection..Infection.was.defined.as.viral.shedding.or.a.≥4-fold.increase.in. NoV.specific.ab.titer..In.total.42/51.volunteers.(82%).were.infected.of.which.13.were. asymptomatic.and.29.symptomatic..When.stratifying.the.groups.by.blood.groups,. Hutson.and.coworkers.(2002).noticed.that.carriers.of.blood.group.O.were.at.highest. risk.of.developing.symptomatic.NoV.infection.while.carries.of.blood.group.B.were.at. significantly.lower.risk.of.symptomatic.infection..Carriers.of.blood.group.O.were.at. lower.risk.of.being.asymptomatically.infected,.as.compared.to.other.blood.groups.. The.authors.concluded.that.also.other.factors.must.be.important.for.susceptibility,. as.the.correlation.to.ABO.blood.groups.was.not.complete,.and.they.also.mentioned. that.secretor.status.may.be.important..

Secretor status and histo-blood group antigens

Secretor.status.describes.the.presence.or.absence.of.ABO(H).histo-blood.groups.in. saliva.and.on.various.mucosal.tissues,.such.as.the.intestine.(Mollicone.et.al..1995;. Ravn.and.Dabelsteen.2000)..Approximately.20%.of.the.Caucasian.population.lack. ABO.in.secretions.and.on.mucosal.epithelia.and.are.called.non-secretors.(secretor-negative). (Koda. et. al.. 2001;. Kindberg. et. al.. 2006;. Larsson. et. al.. 2006;. Kindberg. et. al.. 2007).. The. ABO. antigens. are. carbohydrate. structures. present. as. terminal. epitopes. of. -. and. -linked. glycans. on. glycoproteins. and. on. glycosphingolipids.. The. non-secretor. phenotype. is. caused. by. inactivating. mutations. in. the. . (fucosyltransferase.2).gene.. .encodes.the.enzyme.FucT-II,.one.of.two.active. human.α1,2-fucosyltransferases,.which.adds.a.fucose.in.α1,2.linkage.to.a.terminal. galactose.(Figure. 6).. The. other. one,. FucT-I. is. mainly. expressed. in. progenitors. of. our.red.blood.cells,.and.catalyzes.the.build.up.of.our.ABO.blood.type.on.these.cells.. On.the.contrary. .is.mainly.expressed.in.mucosal.tissues..(Mollicone.et.al..1995;. Ravn.and.Dabelsteen.2000).

Carriers.of.at.least.one.functional. .allele.are.called.secretors.(secretor-positive). and. have. the. ability. to. express. α1,2-fucosylated. type. 1. chain. based. antigens. in. secretions.and. on.mucosal.tissues.. The. typical.acceptors.for. FucT-II. are. the. type. 1. (Galb1,3GlcNAcb1-R). and. type. 3. (Galb1,3GalNAcα1-R). chains. although. FucT-II. is. also.active.towards.the.type.2.chain.(Galb1,4GlcNAcb1-R),.normally.associated.with. FucT-I.(Ravn.and.Dabelsteen.2000)..Depending.on.the.acceptor.chain,.the.product. formed.is.called.H.type.1,.2.or.3,.all.chains.having.the.disaccharide.Fucα1,2-Gal.in. common..The.H.antigens.may.be.further.extended.into.A.or.B.antigens,.by.the.α1,3-GalNAc-.or.α1,3-Gal.transferase.encoded.by.the.blood.group.AB(O).gene,.depending. on. the. active. glycosyltransferases. of. the. individual.. The. completely. dominating. (>99%).inactivating.mutation.in. .among.Caucasians.is.G428A,.which.introduces. a.premature.stop.codon.in.the.gene.(Kelly.et.al..1995)..The.missense.mutation.A385T. causes. a. weakened. enzyme. activity. and. the. so-called. weak. secretor. phenotype,. common.in.Asia.(Yu.et.al..1995;.Henry.et.al..1996a)..A.non-sense.mutation.at.nt.571. (C>T).is.present.mainly.in.Philippine.and.Samoan.populations.(Henry.et.al..1996b;. Koda.et.al..1996;.Peng.et.al..1999;.Pang.et.al..2000). . The.addition.of.an.α1,4-linked.fucose.to.the.subterminal.GlcNAc.of.the.type.1.chain. precursor.forms.the.Lewis.a.(Lea_{).structure,.whereas.a.similar.fucosylation.of.H.type.}

1.forms.Lewis.b.(Leb_{)..Similarly,.A.type.1.and.B.type.1.can.be.transformed.into.ALe}b_.and.

BLeb_{,.respectively.(Figure.6)..The.main.fucosyltransferase.catalyzing.these.reactions.}

is.FucT-III,.encoded.by.the. ( ).gene..Homozygote.carriers.of.inactive. . alleles.lack.Lea_.and.Leb_{.structures..Such.individuals.are.denoted.Lewis-negative.and.}

constitute.about.5%.of.the.Caucasian.population.(Mollison.et.al..1993;.Marionneau.et. al..2001).but.is.more.common.in.for.example.Central.America.(Bucardo.et.al..2009).. Notably,.the.secretor.status.of.Lewis-negative.individuals.and.the.ABO.blood.group. of.secretor-negative.individuals.may.not.be.determined.using.abs.towards.Lea_,.Leb_,. A.or.B.antigens.as.those.individuals.do.not.express.any.of.these.structures.. Figure.6..Schematic.overview.of.the.synthesis.of.Lewis.and.ABH.antigens,.based.in.the.H.type.1. chain..Antigen.names.are. shown.in.bold.and.enzymes.are. in.gray.boxes..Only.secretor-positive. individuals,. carrying. at. least. one. functional. . allele,. express. α1,2-fucosylated. antigens. in. secretions.such.as.saliva.and.on.mucosal.tissues..These.include.the.Leb_{.and.ABH.antigens,.expressed.}

in.different.combinations.depending.on.the.active.glycosyltrasferases.of.the.individual..Secretor-negative.individuals.express.the.H.type.1.precursor.or,.if.Lewis-positive,.the.Lewis.a.antigen..Gal:. Galactose,. GalNAc:. N-Acetylgalactosamine,. Glc:. Glucose,. GlcNAc:. N-Acetylglucosamine.. FucT:. Fucosyltransferase..Lea_{:.Lewis.a,.Le}b_:.Lewis.b.

Secretor.status.has.been.associated.with.increased.or.decreased.susceptibility.to. several.different.infectious.diseases,.such.as.respiratory.bacterial.infections,.urinary. tract.infections.caused.by. .and.infection.with.respiratory.viruses.(Sheinfeld. et.al..1989;.Raza.et.al..1991;.Stapleton.et.al..1995).(Table.2)..In.general,.secretors. seems.to.be.at.higher.risk.for.infection.with.viral.infections,.while.secretor-negative. individuals.seems.more.susceptible.to.infections.with.various.bacterias.

Table. 2.. Reported. associations. between. secretor. status. and. bacterial. and. viral. infections

Pathogen.or. . . Secretor-.. Secretor-. Reference disease.. . . positive. negative.

... . . ...+. . (Blackwell.et.al..1986b) . ... . . ...+. . (Blackwell.et.al..1986a) . . . ...+. . (Blackwell.et.al..1986a) UVI.caused.by. . . . ...+. . (Sheinfeld.et.al..1989;.. . . . . . Stapleton.et.al..1995) Diarrhea.caused.by.ETEC. . . ...+. . (Ahmed.et.al..2009) Influenza.A.and.B.virus. . ...+.. . . . (Raza.et.al..1991) Rhinovirus. . . ...+. . . . (Raza.et.al..1991) Respiratory.syncytial.virus. ...+. . . . (Raza.et.al..1991) Echovirus. . . ...+. . . . (Raza.et.al..1991) HIV.infection... . . ...+. . . . (Blackwell.et.al..1991;.Ali.et.al..2000) HIV.disease.progression.. ...+. . . . (Kindberg.et.al..2006) ETEC:.enterotoxigenic. .

Secretor status and susceptibility to norovirus

Using. an. . model,. Marionneau. and. coworkers. (2002). studied. binding. of. NoV.to.intestinal.epithelia.and.noted.a.difference.in.binding.ability.between.cells. from.different.individuals..They.suggested.that.NoV.infections.may.be.dependent. on.secretor.status.and.the.presence.of.α1,2-fucosylated.type.1.antigens.and.could. show.not.only.that.NoV.bound.only.to.epithelia.from.secretor-positive.individuals. but.also.that.transfection.of.cell.lines.with. .allowed.attachment.of.NoV.VLP.to. otherwise.non-permissive.cells..Following.this.finding,.Lindesmith.and.coworkers. (2003).used.GI.1.NoV.isolate.to.infect.77.volunteers.and.studied.the.outcome.in.terms. of.symptoms,.viral.RNA.in.feces.and.ab.response.after.challenge..34/77.individuals. (44%).were.infected.as.measured.by.a.four-fold.increase.in.ab.titer.as.compared.to. pre-challenge.titer,.or.by.presence.of.NoV.RNA.in.feces..The.secretor.status.and. ABO.blood.group.of.all.individuals.was.determined.and.the.results.showed.that.only. secretor-positive. individuals. were. symptomatically. or. asymptomatically. infected. with.NoV..Moreover,.once.again.using.sera.from.a.previous.volunteer.study.(Graham. et.al..1994).Hutson.and.co-workers.(2005).could,.in.agreement.with.the.data.from. Lindesmith.and.co-workers.(Lindesmith.et.al..2003),.show.that.susceptibility.to.NoV. infection.was.in.complete.association.with.secretor.status..

Studies of authentic norovirus outbreaks

Following. the. volunteer. studies,. several. authentic. outbreaks. have. been. studied. regarding.host.susceptibility.to.NoV.disease..Thorvén.and.co-workers.(2005).studied. three. nosocomial. outbreaks,. caused. by. GII.4. NoV. as. well. as. sporadic. outbreaks.

caused. by. GII.2. and. GI.6. NoV.. They. noticed. a. complete. association. between. symptomatic. infection. and. secretor. status. with. only. secretor-positive. individuals. being.symptomatically.infected.with.NoV..Furthermore,.using.stool.samples.from. one.of.the.hospital.outbreaks,.the.authors.observed.that.the.virus.bound.saliva.only. from.secretor-positive.and.not.secretor-negative.individuals,.indicating.that.the.viral. receptor.was.present.in.saliva.only.from.secretors. Tan.and.co-workers.(2008b).studied.a.total.of.146.individuals.affected.by.outbreaks. with.GII.4.and.GII.3.NoV.in.China.and.observed,.in.agreement.with.Thorvén.and.co-workers.(2005),.a.total.protection.of.secretor-negative.individuals.to.symptomatic. infection..Bucardo.and.co-workers.investigated.several.sporadic.cases.with.different. NoV.genotypes.in.Nicaragua,.and.observed.that.only.secretor-positive.individuals. were.symptomatically.(2009).or.asymptomatically.(2010).infected..The.number.of. samples.with.each.NoV.genotype.was.however.low.and.the.frequency.of.secretor-negative.individuals,.as.determined.by.homozygous.carriage.of.the. .428.allele. or.a.Lea+b-_{.phenotype.in.saliva,.was.only.6%.among.population.controls,.suggesting.}

that. more. studies. are. needed. to. elucidate. the. importance.of. secretor. status. for. susceptibility.to.NoV.in.Central.America.

There.are.however.exceptions.to.the.secretor-dependent.NoV.susceptibility.pattern.. Rockx. and. co-workers. (2005). studied. a. water-borne. NoV. GI.3. outbreak. among. children,.and.noticed.that.four.of.24.infected.individuals.were.secretor-negative.. Viral.RNA.was.detected.in.stool.samples.from.all.four.infected.secretor-negative. children.(Rockx.et.al..2005)..Furthermore,.in.a.challenge.study.using.Snow.Mountain. NoV.(SMV,.GII.2),.one.of.three.secretor-negative.(33%),.and.eight.of.twelve.of.the. secretor-positive.individuals.(67%).were.infected.as.shown.by.detection.of.NoV.RNA. in.stool.(Lindesmith.et.al..2005)..The.studies.by.Rockx.and.co-workers.(2005).and. Lindesmith.and.co-workers.(2005).showed.no.association.between.secretor.status. and.susceptibility.to.NoV,.suggesting.both.that.secretor-negative.individuals.may.be. infected.by.NoV.and.that.different.NoV.genotypes.have.the.ability.to.infect.different. individuals..Rockx.and.co-workers.(2005).also.investigated.the.importance.of.ABO. blood.groups.in.NoV.infections.and.found.that.carriers.of.blood.group.B.were.less. likely.to.develop.disease.compared.to.carriers.of.blood.group.A.and.O..Only.two. carriers.of.blood.group.B.were.however.included.in.the.study,.making.it.difficult. to.interpret.the.importance.of.the.finding..Meyer.and.co-workers.(2004).reported. of.a.NoV.outbreak.in.Germany.where.carriers.of.blood.group.O.were.at.lower.risk. than.other.blood.groups,.to.develop.disease..Fretz.and.co-workers.(2005).found.no. association.to.ABO.blood.groups.in.a.case-control.study.of.sporadic.NoV.outbreaks. in.Switzerland,.and.the.same.was.the.result.in.an.outbreak.study.of.GII.NoV.(Halperin.

et.al..2008)..Today.there.is.no.general.association.between.a.certain.blood.group. and.susceptibility.to.any.NoV.genotype.

Indirect.proof.of.NoV.infection.in.non-secretors.comes.from.a.study.of.titers.and. presence.of.abs.to.NoV.in.healthy.Swedish.blood.donors.(Larsson.et.al..2006)..By. investigating. the. presence. of. anti-GII.4. NoV. IgG. abs. among. 105. Swedish. blood. donors,. Larsson. and. co-workers. (2006). could. show. that. even. though. secretor-positive.individuals.had.significantly.higher.prevalence.and.titers.of.GII.4.NoV-specific. abs. compared. to. secretor-negative. individuals,. most. individuals. were. indeed. ab-positive..As.the.switch.from.IgM.to.IgG.abs.occurs.only.after.infection.(Janeway.et. al..2001),.this.suggests.that.most.individuals.have.at.some.time-point.been.infected. with.GII.4.NoV,.or.a.strain.generating.abs.that.cross-react.with.GII.4.NoV.VLPs..No. difference. in. ab. titer. or. prevalence. was. seen. between. carriers. of. different. ABO. blood.types.or.Lewis.genotypes.(Larsson.et.al..2006)..Rockx.and.co-workers.(2005). investigated.the.association.between.different.ABO.blood.groups.and.the.presence. of.abs. to.GI. and.GII. NoV.. While. no.difference. was. found.for. GII. NoV,.individuals. carrying.blood.group.B.were.less.likely.to.have.abs.to.GI.NoV.

Virus-like particles

The.lack.of.animal.and.cell.culture.models.for.human.NoV.(Duizer.et.al..2004b).has. restricted. the. studies. of. the. virus.. Using. VLPs. it. is. however. possible. to. test. the. binding. specificities. of. different. NoV. strains. to. for. example. saliva. with. different. antigen.content,.and.to.synthetic.substances..VLPs.are.expressed.in.baculovirus.or. yeast.and.have.similar.morphological.and.immunologic.characteristics.as.authentic. NoV.(Jiang.et.al..1992;.Xia.et.al..2007)..It.is.also.possible.to.express.only.the.P-dimer. or.P-particle.(lacking.the.inner.S-domain).in. ,.yeast.or.baculovirus.and.these. show.the.same.binding.characteristics.as.VLPs.(Tan.et.al..2004;.Tan.and.Jiang.2005;. Tan.et.al..2008a)..Huang.and.co-workers.(2003;.2005).analyzed.the.binding-pattern. of. fourteen. different. NoV. VLPs. and. identified. seven. different. binding. patterns,. which.could.be.divided.into.A/B-binding.and.Lewis-binding.patterns.(Huang.et.al.. 2005)..The.A/B-binding.strains.bound.one.or.several.epitopes.of.A,.B.or.H,.while.the. Lewis-binding.strains.recognize.Lewis.(Lea_,.Leb_,.Lex_.and/or.Ley_{).or.H.type.1.antigen..}

While.Lea_.and.Leb.._{are.products.of.an.addition.of.α1-4-linked.fucose.on.the.H.type.1.}

antigen.or.its.precursor.(Figure.6),.Lex._and.Ley_{.are.based.on.the.related.but.different.}

H. type. 2. strain.. Of. fourteen. different. VLPs. investigated,. Huang. and. co-workers. (2003;.2005).found.that.nine.bound.A.or.B.structures,.three.bound.Lewis.epitopes. and.two.NoV.strains.(DSV.and.VA115,.both.GI.3).bound.none.of.the.saliva.samples. or.oligosaccharide.conjugates.tested..Shirato.and.co-workers.(2008).presented.a. similar.study.on.VLP-binding.to.saliva.but.unlike.Huang.and.co-workers.(2003;.2005).

they.did.not.find.any.VLPs.that.bound.only.Lewis.epitopes..Shirato.and.co-workers. detected.no.binding.of.GII.1.and.GII.14.to.any.saliva.or.synthetic.carbohydrate.and. only. weak. binding. of. GII.12. VLP. to. secretor-positive. saliva. from. blood. group. B. carriers,.data.that.does.not.agree.with.that.from.Huang.and.co-workers.(2003;.2005).. While.Huang.and.coworkers.(2003;.2005).detected.no.binding.of.GI.3.VLPs.to.any. of.the.tested.epitopes,.Shirato.and.co-workers.detected.strong.binding.of.a.similar. VLP.to.saliva.from.both.secretors.and.weak.secretors.as.well.as.to.several.synthetic. glycoconjugates..The.discrepancies.between.the.results.of.the.VLP.binding.studies. may. be. due. to. small. genetic. differences. between. strains,. affecting. the. binding. properties.of.the.VLPs..Moreover,.instead.of.secretor-negative.individuals,.the.study. by.Shirato.and.co-workers.(2008).included.weak.secretors,.i.e..homozygous.carriers. of.the.missense.mutation.385.in. ,.which.probably.affected.the.results..

While. NoV-saliva. ELISA. assays. have. been. instrumental. in. investigating. virus-receptor.interactions.(Huang.et.al..2005;.Tan.and.Jiang.2007;.Shirato.et.al..2008),. the.clinical.significance.of. .binding.characteristics.needs.to.be.further.verified. in. authentic. outbreaks. and. sporadic. cases.. Norwalk. virus. is. the. most. frequently. used.strain.in.volunteer.studies,.and.while.the.virus.can.infect.individuals.of.blood. group.B.(Hutson.et.al..2002;.Lindesmith.et.al..2003).some.studies.have.shown.that.it. does.not.bind.to.saliva.from.blood.group.B.donors.(Harrington.et.al..2002;.Huang.et. al..2005)..In.the.study.by.Shirato.an.co-workers.(2008),.GI.1.virus.did.however.bind. to.saliva.from.carriers.of.blood.group.B,.although.not.as.strong.as.to.saliva.from. carriers.of.blood.group.A,.AB.and.O..GI.1.NoV.was.also.able.to.agglutinate.human. red.blood.cells.from.carriers.of.all.blood.group,.although.only.in.4/14.samples.from. blood.group.B.carriers.compared.to.11/11.from.blood.group.O.and.9/9.blood.group. A.carriers.(Hutson.et.al..2003)..Furthermore,.while.volunteer.challenge.studies.with. the. SMV. norovirus. (GII.2). revealed. infection. of. carriers. of. all. ABO. blood. groups. (Lindesmith. et. al.. 2005),. the. virus. did. not. bind. to. saliva. from. secretor-positive. individuals.with.blood.group.A.or.O..(Harrington.et.al..2002)..Moreover,.while.SMV. infected. non-secretors. after. challenge. (Lindesmith. et. al.. 2005). it. did. not. bind. to. saliva. from. non-secretors. (Harrington. et. al.. 2002),. suggesting. the. existence. of. other.HBGA.receptors,.as.Lea .seems.not.to.be.the.determinant.for.infection.in.non-secretors..These.data.show.that.it.is.important.to.complement.VLP-binding.studies. and.genetic/phenotypic.screening.for.susceptibility.with.authentic.virus.binding.data. to.identify.the.real.receptor.of.NoV..Few.studies.have.tried.to.use.authentic.virus.to. elucidate.binding.to.saliva.or.synthetic.carbohydrates..Harrington.and.co-workers. (2004).used.biotinylated.HBGA.for.binding.of.NoV.in.clinical.stool.samples..With.the. help.of.magnetic.beads.conjugated.with.streptavidin,.they.were.able.to.determine. specific. binding. of. different. NoV. strains. to. different. HBGA.. In. concordance. with.

the.VLP.binding.data.of.Huang.and.co-workers.(2005),.Harrington.and.co-workers. (2004).did.not.detect.binding.of.GI.3.virus.to.any.of.the.HBGA.conjugates.tested.. Furthermore,.they.also.found.that.binding.of.VLPs.to.HBGA.increased.in.the.presence. of. 1%. stool. suspension,. suggesting. that. some. component. in. stool. enhances. the. binding.capacity.of.the.virus.(Harrington.et.al..2004)..As.mentioned.above,.Thorvén. and.co-workers.(2005).tested.binding.of.authentic.NoV.to.saliva.samples.and.found. binding.to.secretor-positive.but.not.secretor-negative.saliva..These.samples.were,. however,.not.classified.regarding.ABO.or.Lewis.antigens.

Most.studies.have.investigated.and.revealed.binding.of.NoV.to.fucosylated.type. 1.chain.conjugates,.including.Lea_.and.Leb_{.as.well.as.to.A,.B.and.H.(Harrington.et.al..}

2002;.Lindesmith.et.al..2003;.Huang.et.al..2005;.Shirato.et.al..2008),.but.Rydell.and. co-workers.(2009).found.that.GII.3.and.GII.4,.but.not.GI.1.VLPs.bound.also.sialyated. type. 2. chain. conjugates,. such. as. SLex_{.. They. found. that. binding. was.}

secretor-dependent.and.detected.binding.only.to.saliva.from.secretor-positive.individuals.. The.VLPs.did.not.recognize.SLex_{. -linked.to.the.backbone.of.mucins.in.saliva.but.}

only.HSA-conjugated.SLex_{.(Rydell.et.al..2009)..Sialylated.receptors.are.widely.used.}

by.different.viruses.(Olofsson.and.Bergstrom.2005).and.it.is.possible.that.certain. NoV.strains.could.use.sialylated.structures.as.receptors.or.co-receptors..

Immunity and viral evolution

Despite.attempts.to.elucidate.what.components.of.the.immune.system.that.are. the.most.important.to.combat.a.NoV.infection.very.little.is.known.so.far..A.few. early. studies. (Black. et. al.. 1982;. Ryder. et. al.. 1985). suggested. that. NoV. infection. initiate.a.long-term.protective.immune.response,.but.volunteer.studies.have.shown. that.individuals.can.be.re-infected.with.the.exact.same.NoV.strain.(Parrino.et.al.. 1977;.Johnson.et.al..1990)..A.short-term.protection.seems.however.to.be.present. after.challenge.(Parrino.et.al..1977;.Johnson.et.al..1990)..Using.three.different.NoV. strains.(two.GI.and.one.GII.strain),.Wyatt.and.co-workers.(1974).noted.that.while.a. homologous.immunity.developed.after.challenge,.no.heterologous.immunity.was. seen.between.GI.and.GII.NoV..This.agrees.with.data.from.Lindesmith.and.coworkers. (2005).who.also.noted.the.development.of.a.homologous.humoral.immune.response.. Studies.using.acute.and.convalescent.sera.have.suggested.that.GII.4.have.the.ability. to.escape.immunity.through.epochal.evolution.(Siebenga.et.al..2007b;.Lindesmith. et.al..2008)..Lindesmith.and.co-workers.(2008).compared.the.binding.pattern.of. different.GII.4.NoV.and.noted.that.small.changes.in.the.P2.region.enabled.the.virus. to.change.its.binding.specificity,.at.least.to.some.degree..One.GII.4.could.even.bind. to.secretor-negative.saliva,.although.only.at.37°C..The.change.in.the.P2.region.does.

not.only.affect.the.binding.ability.but.it.also.enables.the.virus.to.evade.immunity. (Lindesmith.et.al..2008)..This.may.explain.why.GII.4.can.persist.in.the.population. despite. the. fact. that. the. same. genotype. has. been. circulating. for. more. than. a. decade..It.is.possible.that.immune.evasion.occur.also.in.other.genotypes,.such.as. GII.3.(Nilsson.et.al..2003;.Siebenga.et.al..2008),.while.data.have.suggested.that.GI. NoV.are.more.structurally.stable.(Choi.et.al..2008).and.that.the.immune.response. elicited.to.GI.NoV.is.broader.than.that.to.GII.NoV.(Lindesmith.et.al..2010).. Attempts.have.been.made.to.elucidate.the.position.in.the.viral.capsid,.involved.in. binding.to.the.target.cell..Suggestions.of.important.aa.for.binding.to.HBGA.come. from.high.resolution.methods:.Choi.and.co-workers.(2008).found.a.tryptophane.at. aa.375.to.be.important.for.hydrophobic.interaction.with.the.A.and.H.type.1.structure.. and. X-ray. crystallography. studies. of. P-dimers. in. complex. with. α1,2-fucosylated. glycans.have.suggested.that.the.Norwalk.and.the.VA387.(GII.4).strains.use.closely. located,.but.distinct,.binding.sites.(Cao.et.al..2007b;.Bu.et.al..2008;.Choi.et.al..2008).. The.aa.expected.to.be.involved.in.HBGA.binding.have.also.been.confirmed.in.a.study. using.site-directed.mutagenesis,.but.this.study.also.showed.that.aa.not.expected.to. be.in.direct.contact.with.the.glycans.were.required.for.binding.(Tan.et.al..2008c). Comparison.of.the.ORF2.from.66.GII.4.strains.showed.a.20%.diversity.in.the.hyper-variable.P2.region,.and.also.revealed.several.evolutionary.hot.spots,.of.which.six.(aa. 296-298,.“site.A”.and.aa.393-395,.“site.B”).had.impact.on.the.biochemical.properties. of.the.capsid.(Allen.et.al..2008)..Both.site.A.and.site.B.are.found.in.the.protruding. P2.domain.and.site.A.is.situated.close.to.the.predicted.HBGA.binding.site.(Cao.et. al..2007a;.Allen.et.al..2008).and.may.be.of.interest.for.the.understanding.of.NoV. evolution.and.immunity...

Treatment and preventive measures

There.is.no.special.treatment.available.for.NoV.infections.but.hospitalized.patients. can. receive. oral. or. intravenous. rehydration.. As. described. above,. the. number. of. fatalities.is.high.in.developing.countries.and.although.the.disease.is.often.considered. relatively. mild. in. the. western. world,. with. few. severe. cases. (Meeroff. et. al.. 1980;. Patel.et.al..2008),.the.high.patient.number.makes.it.costly.for.the.society.(Lopman. et.al..2004b)..Due.to.the.antigenic.similarity.between.authentic.virus.and.VLPs.or. P-particles,.these.have.been.used.in.the.trials.of.developing.a.vaccine.(Chachu.et.al.. 2008;.Tan.et.al..2008a;.LoBue.et.al..2009;.Donaldson.et.al..2010;.Herbst-Kralovetz. et.al..2010)..However,.as.even.an.authentic.infection.does.not.leave.the.patients. resistant.to.a.second.infection.with.the.same.isolate.(Parrino.et.al..1977;.Johnson.et. al..1990).the.development.of.a.vaccine.will.be.a.very.challenging.task...

Tick-borne encephalitis virus

While. herpesvirus. is. the. most. common. cause. of. acute. sporadic. encephalitis. (Tyler.2004),.TBEV.is,.after.Japanese.encephalitis.virus,.the.second.most.common. neurotropic.flavivirus.causing.disease,.with.10000.cases.every.year.(Kunz.and.Heinz. 2003).. In. Europe,. 3000. patients. are. hospitalized. and. treated. for. TBE. each. year. (WHO/EURO.2004)..TBEV.was.the.most.common.cause.of.viral.meningoencephalitis. in. Sweden. 2008. (SMI. 2008).. According. to. data. from. the. Swedish. institute. for. infetious.disease.control.(SMI).(Hjertqvist.2010),.the.mean.TBE.incidence.in.Swedish. population. between. 2000. and. 2009. has. been. approximately. 150. cases/year.. The. number. has. almost. doubled. since. the. 1990’s. with. the. highest. annual. reported. number.being.224,.in.2008..The.increased.incidence.may.be.due.to.several.factors. such. as. increased. awareness. among. patients. and. physicians. and. changed. habits. of.the.population.(Hjertqvist.2010)..The.peak.incidence.of.reported.TBE.occur.at. seasonal.peaks.of.feeding.and.activity.of.the.ticks.(Gritsun.et.al..2003a;.Kerbo.et.al.. 2005a). The.disease.TBE.was.first.described.in.Austria,.in.the.early.1930’s.(Schneider.1931).as. a.disease.of.unknown.etiology,.which.reappeared.yearly.during.spring.and.summer.. In.the.coming.years,.reports.of.the.disease.came.from.the.former.Soviet.Union.and. in.the.1940’.the.disease.was.seen.on.Åland.and.in.former.Czechoslovakia..In.1950’s,. Holmgren.and.co-workers.(1959).confirmed.the.first.Swedish.case.of.TBE..The.first. isolation.of.European.TBEV.took.place.in.former.Czechoslovakia.in.1948,.during.an. epidemic.(Burke.and.Monath.2001). TBE.has.several.synonyms,.such.as.the.Russian.spring-summer.encephalitis.(RSSE),. Kumlinge.disease,.Central.European.encephalitis,.Taiga.encephalitis,.Diphasic.milk. fever. and. Früsommer-Meningoenzephalitis. (FSME). (Kunz. and. Heinz. 2003).. The. disease.is.spread.mainly.from.ticks,.via.tick.saliva,.often.early.after.a.tick.bite,.as. the.tick.suck.blood..Ticks.are.obligate,.blood-sucking,.small.arthropods.that.feed. on.mammals,.birds,.reptiles.and.amphibians.all.over.the.world..They.belong.to.the. superfamily.Ixodoidea.in.the.subclass.Acarina..The.external.body.of.the.tick.consists. of.legs,.the.capitulum,.the.body.and.the.holdfast.organ,.called.the.hyptosome..Ticks. pass.trough.three.stages.of.life;.from.larvae.via.nymphs.to.adult.ticks..While.nymphs. and.adult.ticks.have.eight.legs,.larval.ticks.have.only.six..To.pass.from.one.life.stage.to. another,.the.ticks.have.to.take.a.blood.meal..(Anderson.and.Magnarelli.2008).TBEV. is.spread.between.ticks.both.vertically.(from.female.to.egg).(Bakhvalova.et.al..2006). and.horizontally.(between.ticks.co-feeding.on.the.same.animal)..Horizontal.spread. does.not.demand.viremia,.since.virus.can.be.spread.locally.in.the.tissue.(Labuda. et.al..1996)..As.a.tick.becomes.infected.with.TBEV.the.infection.is.permanent,.but.

asymptomatic.. The. tick. is. both. the. natural. host. and. the. vector. of. the. virus,. but. the.virus.circulates.between.the.ticks.and.various.wild.vertebrate.such.as.rodents. and.deer.(Burke.and.Monath.2001)..Vertical.transmission.has.also.been.shown.in. mammal.hosts.(Bakhvalova.et.al..2009).

Genome structure and classification of tick-borne encephalitis virus

TBEV.belong.to.the.genus.flavivirus.in.the.family. ..The.word.flavus.means. yellow.in.Latin.and.the.virus.family.got.its.name.from.the.prototype.yellow.fever.virus,. which.cause.liver.damage.and.jaundice.and.hence.a.yellowish.skin..Other.viruses.of. the.same.genera.are.Japanese.encephalitis.virus.and.West.Nile.virus.(WNV)..TBEV. belongs.to.the.tick-borne.flavivirus.group..The.other.two.groups.are.the.mosquito-borne.and.the.group.of.virus.with.no.known.vector..The.tick-borne.group.is.further. divided.into.the.mammalian.(including.TBEV).and.the.seabird.group..All.flaviviruses. share. very. similar. structural. features. as. well. as. the. organization.of. the. genome.. (Lindenbach.2001).The.species.TBEV.was,.based.on.serological.analyses.first.divided. into.two.subgroups.(Clarke.1964).and.a.third.was.later.identified.by.phylogenetic. analyses. (Gritsun. et. al.. 1993).. The. three. subtypes. have. later. been. confirmed. by. sequencing. analysis. (Ecker. et. al.. 1999). and. they. are. today. called. Far. Eastern. (previously.known.as.RSSE),.Siberian.(previously.west-Siberian).and.the.European. (previously.Central.European.Encephalitis).subtype.. Figure.7..Organization.of.the.TBEV.genome..The.genome.is.a.ssRNA.(approximately.11.kb).with. positive.polarity.and.consist.of.a.single.ORF.encoding.a.polyprotein..The.polyprotein.is.cleaved.by. host.and.viral.proteases.into.three.structural.(C,.M.and.E).and.seven.non-structural.(NS).proteins.. The.TBEV.RNA.is.capped.in.the.5´.end.but.unlike.NoV.and.human.RNA,.the.TBEV.RNA.has.a.3´-hydroxyl. group,.instead.of.a.poly.A.tail.

TBEV. are. enveloped. viruses. of. approximately. 50. nm. (Gritsun. et. al.. 2003b). with. a. ssRNA. genome. of. approximately. 11. kb,. encoding. around. 3000. aa. (Figure. 7). (Lindenbach.2001)..The.genome.consist.of.one.single.ORF.which.is.translated.into.a. polyprotein,.later.cleaved.by.host.and.virus.proteases.(Lindenbach.2001)..The.viral. particle. contains. three. structural. proteins;. the. major. E. (envelope). protein. in. the. membrane,.the.small.M.(membrane).protein.and.the.C.(capsid).protein.(Figure.8). and.the.capsid.has.a.T=3.structure.(Heinz.2003)..The.M.protein.is.only.found.in.mature. virions.(Pletnev.et.al..1986)..The.E.protein.is.involved.in.receptor.binding.to.the.host. cell.and.mediates.fusion.of.the.viral.membrane.with.the.host.endosomal.membrane. (Mukhopadhyay. et. al.. 2005).. The. genome. also. encodes. seven. non-structural. proteins,.including.the.RdRp,.a.protease.and.a.helicase.(Lindenbach.2001)..The.virus.

is.relatively.stable.for.being.an.RNA.virus.and.the.difference.of.the.E-protein.within. each.subtype.is.only.about.2%.at.amino.acid.level.and.between.subtypes.less.than. 6%..The.difference.at.the.nt.level.is.almost.17%,.suggesting.that.a.selective.pressure. have.conserved.the.aa.sequence.of.the.E-protein.(Ecker.et.al..1999).

Figure. 8.. Schematic. overview. of. the. TBEV. particle.. The. virus. is. enveloped. and. contains. three. structural.proteins:.the.E.(envelope),.M.(membrane).and.C.(capsid).proteins..(Viralzone,.http:// www.expasy.ch/viralzone.Swiss.Institute.of.Bioinformatics,.with.permission).

The.ticks.spreading.TBEV.are.the. and.the .. .

is.mainly.present.in.Europe.while. .is.found.in.the.Far.East,.Siberia.and. in. the. Ural. region. (Figure. 9).. Hence,. the. European. TBEV. subtype. is. spread. by.

..TBE.is.a.notifiable.disease.in.16.countries.in.Europe.(Figure.10).(Suss.2008). and.in.large.areas.in.Asia,.as.far.east.as.China.and.Japan.and.studies.have.shown. that.approximately.0.1-3%.of. .ticks.in.European.focus.areas.are.carriers.of. TBEV.(Han.et.al..2002;.Suss.et.al..2002;.Han.et.al..2005;.Skarpaas.et.al..2006;.Brinkley. et.al..2008;.D’Agaro.et.al..2009;.Klaus.et.al..2010).although.higher.numbers.have.on. occasion.been.reported.(Casati.et.al..2006)..TBEV.may.at.times.also.be.spread.via.the. alimentary.route,.from.unpasteurized.milk.(Kerbo.et.al..2005b;.Balogh.et.al..2010). or.cheese.(Holzmann.et.al..2009)..In.animals,.the.infection.remains.asymptomatic. and.is.followed.by.full.recovery..The.virus.is.however.secreted.in.the.milk.in.low. concentrations.during.the.viremic.phase,.which.can.make.milk.and.dairy.products. from.unpasteurized.milk.infectious.(Zeman.et.al..2004).

Figure.9..Geographical.distribution.of.Ixodes.ricinus.and.Ixodes.persulcatus..The.distributions.of. the.ticks.are.overlapping.in.the.Baltic.States.and.parts.of.Russia..TBE.endemic.areas.are.found. within.the.dotted.line..(Modified.from.Lindquist.and.Vapalahti.2008,.with.permission).

Symptoms and diagnosis

After.the.tick.bite,.the.virus.replicates.in.the.Langerhans.cells.in.the.skin.and.later. on.reaches.the.lymph.nodes.via.the.lymphatic.system.(Haglund.and.Gunther.2003).. The.virus.is.spread.via.the.blood.stream.and.infects.several.organs,.including.the. reticulo-endotelial.system.(Haglund.and.Gunther.2003)..Serological.screenings.have. suggested.that.70-95%.of.all.TBEV.infections.are.asymptomatic.or.sub-clinical.(Gritsun. et.al..2003b)..The.incubation.time.of.symptomatic.infections.is.generally.1-2.weeks. (Kaiser.1999).but.can.be.up.to.four.weeks.(Gunther.et.al..1997a).and.the.typical. acute.symptoms.are.fever,.head-.and.neckache,.muscle.pain.and.nausea..Viremia. is. present. during. the. acute. phase. and. this. is. often. followed. by. a. symptom. free. period.of.approximately.eight.days.(Kaiser.1999)..Virus.invades.the.CNS.by.passing. the.blood-brain.barrier.(bbb).and.20-30%.of.symptomatic.patients.will.experience.a. second.phase.including.symptoms.such.as.meningitis.or.encephalitis.with.or.without. myelitis.(Kaiser.1999;.Haglund.and.Gunther.2003)..The.disease.can.be.stratified.after. severity.of.symptoms.as.mild,.moderate.or.severe..Mild.cases.include.patients.with. only.meningeal.symptoms,.while.moderate.and.severely.ill.patients.have.mono-.and. multifocal. encephalitis. respectively. (Gunther. et. al.. 1997a).. The. fatality. rate. from. the.European.TBE.is.0.7-2%.(Kaiser.1999;.Ozdemir.et.al..1999).but.many.patients,. almost.40%,.suffer.from.long-term.sequelae,.including.general.weakness,.headache,. tinnitus,.irritability.and.photophobia.as.well.as.memory.disturbances.(Haglund.et. al..1996;.Kaiser.1999;.Laursen.and.Knudsen.2003;.Schmolck.et.al..2005;.Pogorzelski. et. al.. 2006).. Residual. spinal. nerve. paralysis. may. also. occur. (Haglund. et. al.. 1996;. Gunther.et.al..1997a)..The.risk.of.TBE.increase.with.age.and.men.are.at.higher.risk.

than. women. (Holmgren. and. Forsgren. 1990;. Kaiser. 1996;. Gunther. et. al.. 1997a;. Wahlberg.et.al..2006). Figure.10..Areas.endemic.for.the.European.TBEV.subtype.are.marked.in.black..This.subtype.is.mainly. found.in.Central.and.Eastern.Europe,.the.Baltic.states,.on.Åland,.on.the.Swedish.east.coast.around. Stockholm,.east.coast.south.of.Borlänge,.in.Skåne.and.around.lake.Vättern.and.Vänern..(Modified. from.Baxter.Medical.AB,.with.permission). While.TBE.spread.via.tick.bites.occur.randomly,.ingestion.of.infected.milk.may.cause. small. epidemics. in. families. or. villages. (Gresikova. et. al.. 1975;. Zeman. et. al.. 2004;. Holzmann.et.al..2009;.Kriz.et.al..2009;.Balogh.et.al..2010)..Ingestion.of.infectious. TBEV.can.cause.biphasic.milk.fever.(Gritsun.et.al..2003b),.starting.with.the.typical. symptoms.seen.in.TBE.caused.by.tick.bites.and.likewise.followed.by.an.asymptomatic. phase..However,.the.second.phase.of.biphasic.milk.fever,.which.is.present.in.most. cases,.does.not.include.paresis.or.paralysis,.even.though.meningeal.symptoms.may. occur..Patients.almost.always.reach.full.recovery.after.TBE.spread.via.the.alimentary. route..

As.the.symptoms.of.TBE.are.not.specific.for.only.a.TBEV.infection,.the.diagnosis. can. only. be. established. using. laboratory. techniques.. The. method. used. is. often. detection.of.TBEV.specific.IgM.and.IgG.antibodies.by.ELISA,.as.these.are.normally. present. in. all. hospitalized. cases. (Holzmann. 2003).. Detection. of. viral. RNA. is. not. used.for.diagnostics,.as.viremia.has.normally.passed.at.the.time.of.hospitalization. (Holzmann. 2003).. Presence. of. IgG. indicates. native. disease. or. a. history. of. TBE. vaccination,.discussed.later..Detection.of.IgM.in.sera.is.used.for.early.diagnostics. and.a.majority.of.patients.are.IgM-positive.at.time.of.the.beginning.of.the.second. phase.of.disease.(Holzmann.2003;.Venturi.et.al..2009)..This.is.followed.by.a.rise.in. IgG.and.IgA.(Gunther.et.al..1997b)..A.m-capture.ELISA.may.be.used.for.detection.of. intrathecal.IgM.abs.(Gunther.et.al..1997a)..As.TBEV.abs.may.cross-react.with.other. flaviviruses,.neutralization.tests.may.be.needed.to.confirm.ELISA.results.(Gunther. and.Haglund.2005).

Immune response and pathogenesis

When.an.individual.is.infected.with.TBEV.or.vaccinated.with.inactivated.TBEV.vaccine,. a.strong.immune.response.against.TBE.is.elicited.and.in.the.early.phase.an.anti-TBE. IgM.titer.is.almost.always.seen.(Venturi.et.al..2009)..This.response.of.neutralizing.abs,. mainly.against.the.E-protein.(Venturi.et.al..2009),.normally.protect.individuals.from. a.subsequent.infection.(Kaiser.1996),.although.vaccine.breakthroughs.do.occur,.as. will.be.discussed.later..Apart.from.abs,.a.brisk.innate.immune.response,.including. type.I.interferon.(IFN).and.IFN.stimulated.genes.is.important.for.protection.against. TBE.(Robertson.et.al..2009)..As.described.below,.CD8+.T.cells.have.been.suggested. to.be.involved.in.the.pathogenesis.of.TBE.(Ruzek.et.al..2009). The.pathogenesis.behind.the.development.of.encephalitis.after.a.flavivirus.infection. is.still.far.from.clear..It.has.been.suggested.that.it.may.be.due.to.either.direct.neuronal. damage.by.the.virus,.a.virus-induced.inflammatory.response.or.a.combination.of. both.(Chambers.and.Diamond.2003;.King.et.al..2007)..In.a.recent.study.by.Ruzek. and.co-workers.(2009).it.was.shown.that.mice.lacking.CD8+.T.cells.as.well.as.SCID. mice,.lacking.T.and.B.cells,.survive.a.longer.time.after.subcutaneous.infection.with. TBEV,. as. compared. to. wild-type. (wt). Balb/c. and. C57Bl/6. mice.. The. survival. was. also.shortened.in.SCID.mice.after.adoptive.transfer.of.CD8+.T.cells.suggesting.an. immunopathogenic.role.of.cytotoxic.T.cells.in.TBE.infection..Transfer.of.immune. CD8+.cells.decreased.survival.time.more.than.transfer.of.naïve.cells,.although.the. difference.was.not.significant..On.the.contrary,.adoptive.transfer.of.naïve.CD4+.cells. to.SCID.mice.prolonged.the.survival.time.of.the.SCID.mice,.suggesting.a.protective. role.of.Th.cells.(Ruzek.et.al..2009)..The.viremia.in.spleen.and.blood.was.significantly. higher.in.SCID.mice.compared.to.Balb/c,.C57Bl/6.and.CD8-negative.mice,.and.in.brain.

compared.to.Balb/c.mice.(Ruzek.et.al..2009),.showing.that.the.level.of.viremia.is. not.the.determinant.of.time.of.survival..Hayasaka.and.co-workers.(2009).used.a. mouse.model.to.study.the.immune.response.after.subcutaneous.infection.with.high. and.low.dose.of.TBEV..While.the.rate.of.encephalitis.was.approximately.the.same. despite. the. challenge. dose,. the. fatality. rate. was. higher. (>80%. vs.. 50%). after. the. higher.dose..The.study.showed.that.after.infection.with.the.lower.dose,.the.virus. titer.found.in.the.brain.did.not.differ.between.recovering.and.dying.mice,.but.dying. mice.had.an.increased.level.of.the.potent.pro-inflammatory.cytokine.(Horiuchi.et.al.. 2010).tumor.necrosis.factor.α.(TNFα).as.well.as.a.higher.stress.response.compared. to.mice.recovering.from.encephalitis.(Hayasaka.et.al..2009)..The.authors.conclude. that.a.systemic.inflammatory.response,.as.well.as.the.stress.response.contributes. to.the.outcome.of.TBEV.infection.and.that.mortality.is.the.outcome.of.a.higher.level. of.systemic.inflammation.and.stress. Gelpi.and.co-workers.(2006).studied.the.inflammatory.response.in.human.TBE,.by. using. post-mortem. brain. tissue.. They. found. that. granzym. B-releasing. cytotoxic. T-cells.caused.tissue.destruction.in.TBE.patient.but.also.that.macrophages/microglia. contributed.to.the.pathogenesis..While.B.cells.were.mainly.found.in.the.perivascular. compartment,. IgG. was. found. in. the. brain. parenchyma. in. most. of. the. 26. brains. investigated..Neuronal.apoptosis.was.only.rarely.seen.and.as.several.TBEV.infected. intact.neurons.were.found,.virus.induced.lysis.does.not.seem.to.be.the.cause.of.the. injury.(Gelpi.et.al..2006)..

Antibody-dependent. enhanced. infectivity. have. also. been. reported. but. the. importance.of.this.needs.to.be.further.investigated.(Phillpotts.et.al..1985).

Vaccination against tick-borne encephalitis virus

While.no.specific.treatment.is.present.for.TBE,.there.are.two.registered.vaccines. against.TBEV.infection,.on.the.European.market;.Encepur.produced.by.Novartis.and. FSME-IMMUN.produced.by.Baxter.vaccines.(Lindquist.and.Vapalahti.2008;.Demicheli. et. al.. 2009).. Both. Encepur. and. FSME-IMMUN. are. formaldehyde. inactivated. and. are.given.in.similar.schedules.with.three.intramuscularly.injected.doses.within.the. first.year,.followed.by.boosters.after.first.three.and.then.five.years.(Lindquist.and. Vapalahti.2008)..The.protection.level.has.been.estimated.to.be.approximately.95-99%.(Heinz.et.al..2007).but.vaccine.breakthroughs.are.however.reported.(Gunther.et. al..1997b;.Bender.et.al..2004;.Kleiter.et.al..2007;.Lotric-Furlan.et.al..2008;.Andersson. et.al..2010)..In.a.recent.retrospective.study.27.patients.with.TBE.despite.at.least. two.vaccine.doses.were.investigated.regarding.the.serum.profile.of.TBE.specific.abs. (Andersson.et.al..2010)..Of.these.70%.were.classified.as.having.moderate.or.severe.